Light sheet fluorescence microscopy or selective plane illumination microscopy (SPIM) technology typically relies on illuminating of a specimen in thin optical slices, formed from laser light, exciting the fluorophores in the specimen and acquiring light emitted by the illuminated plane inside the specimen. The direction in which the light is detected is typically perpendicular to the illuminated plane. The resolution of SPIM is often limited by the shape and properties of the light sheet illuminating the specimen.
With SPIM, the lateral resolution is determined by the detection objective lens and the axial resolution is related to the numerical aperture (NA) of the illumination objective. With higher NAs, the axial resolution is similar to confocal fluorescence microscopes. Images from light sheet microscopes exhibit a better signal-to-noise (S/N) ratio and a higher dynamic range than images produced by confocal fluorescence microscopes. With low NAs, the axial resolution is determined by the thickness of the light sheet at its thinnest point (i.e., the waist), with an excellent isotropic Point Spread Function. However, while the axial resolution increases only linearly with increasing NA, the field of view (i.e., FOV) with optimal axial resolution decreases with the square of the NA. This relation results in a fundamental design problem, where a large FOV is not compatible with high axial resolution. Therefore, there is a need for an illumination system that produces the thinnest possible light sheet illumination over the largest possible field of view.